The Impact of Endotoxin on Cell Editing Efficiency: A Comparative Study of 293T and A549 Cells
RNA synthesis has been widely applied in gene editing, vaccine development, and disease treatment. However, the RNA synthesis process may result in residual endotoxins. To investigate whether endotoxin affects cell editing efficiency, our company conducted the following experiment.
Methods
We selected two commonly used cell lines: 293T cells and A549 cells.
- 293T cells are human embryonic kidney cells frequently used in gene expression and virus packaging studies.
- A549 cells are human lung cancer cells commonly employed in cancer research.
In the experiment, B2M sgRNA was introduced into both cell lines using liposome-mediated transfection. We evaluated the impact of endotoxin by measuring the knockout efficiency. The detection limit for endotoxin was set at 0.125 EU/ml to ensure the sensitivity and accuracy of the experiment. A strict detection protocol was followed to guarantee the reliability of the results.
Results
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Comparison of Knockout Efficiency:
In 293T cells, the knockout efficiency of B2M sgRNA was significantly higher than that in A549 cells. This result suggests that 293T cells may have a higher sensitivity and efficiency in gene editing. -
Effect of Endotoxin:
Overall detection results indicate that endotoxin has little impact on transfection efficiency. Even at the detection limit of 0.125 EU/ml, the presence of endotoxin did not significantly reduce the knockout efficiency. This finding provides important insight, suggesting that residual endotoxin during RNA synthesis may not have a significant impact on cell editing efficiency.
Discussion
The experimental results demonstrate that 293T cells exhibit higher gene editing efficiency, which may be closely related to their unique cellular characteristics. Moreover, the presence of endotoxin has only a minimal impact on the editing efficiency of these cells. In other words, trace amounts of endotoxin remaining during sgRNA synthesis do not significantly affect the editing efficiency in cell lines such as 293T and A549. In practical experiments, if low editing efficiency is observed, greater attention should be paid to whether the selection of the N20 sequence is appropriate. It is important to note that this experiment was conducted using common cell lines; therefore, the results may not be generalizable to other cell types (e.g., iPSCs, primary T cells, etc.).
Moving forward, we will further investigate the effects of endotoxin on various cell lines and with different gene editing tools. Our goal is to continually optimize RNA synthesis processes, reduce endotoxin contamination, and explore the applications of additional cell lines and gene editing technologies.
About Pixel Bio
Pixel Bio is a biopharmaceutical company specializing in RNA synthesis. Please feel free to contact us for RNA synthesis services.
Disclaimer:
The content of this article is for reference only and does not constitute any
medical or scientific advice. Please adhere to the relevant standards and
guidelines for specific experimental designs and procedures.
